From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 1991 78: 132-140 Purification and characterization of factor VII 304-Gln: a variant molecule with reduced activity isolated from a clinically unaffected male DP O'Brien, KM Gale, JS Anderson, JH McVey, GJ Miller, TW Meade and EG Tuddenham Updated information and services can be found at: http://www.bloodjournal.org/content/78/1/132.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on October 28, 2014. For personal use only. Purification and Characterization of Factor VI1 304-Gln: A Variant Molecule With Reduced Activity Isolated From a Clinically Unaffected Male By Donogh P. O’Brien, Karen M. Gale, J. Simon Anderson, John H. McVey, George J. Miller, Tom W. Meade, and Edward G.D. Tuddenham Factor VI1 (WII) is the plasma serine protease zymogen which, on binding to its cellular receptor tissue factor (TF), initiates blood coagulation.A 47-year-old man with no clinical bleeding tendency was found to have undetectable plasma FVll activity when tested in a one-stage assay using rabbit brain TF, but 0.3 U/mL using recombinant human TF and 1.04 U/mL FVll antigen. Variant FVll purified from his plasma showed an identical migration on sodium dodecyl sulfate-polyacrylamidegel electrophoresis to wild-type zymogen. By enzyme kinetic analysis the Km of the variant using FX as a substrate was 12-fold higher than that of F ACTOR VI1 (FVII) is a serine protease of the extrinsic blood coagulation cascade. It belongs to the vitamin K-dependent group of serine proteases, with homology to FIX, FX, prothrombin, and protein C. The single-chain zymogen has a relative molecular mass of 53 Kd that is converted to a disulphide-linked two-chain enzyme (FVIIa) after proteolysis of an internal arginine-isoleucine bond.’ This activation is effected by FXa, FIXa, and thrombin in vitro. FVIIa has little catalytic activity in the absence of an essential cofactor, tissue factor (TF), a cellular receptor constitutively expressed by many cells of the subendothelium. FVII and FVIIa form a 1:l stoichiometric complex with TF2 and this complex rapidly converts zymogen FX and FIX to the active enzymes. The formation of a complex between TF and FVII is widely thought to represent the primary stimulus for blood coagulation.’ In this study we describe the biochemical analysis of a dysfunctional FVII molecule isolated from the plasma of a clinically unaffected male with normal FV1I:Ag (1.04 U FVII:Ag/mL) but with reduced function (FVI1:C 0.3 U/mL). Thus, this molecule can be described as cross-reacting material positive (CRM+). We have developed a micropurification scheme for the isolation of highly purified FVII from small donations of plasma to facilitate the analysis of the specific defect in this protein. Our results suggest that the defect is the consequence of a point mutation in the FVII gene resulting in a radical substitution in the catalytic domain of the molecule, which most probably affects its interaction with TF. From the Haemostasis Research Group, MRC Clinical Research Centre; and MRC Epidemiology and Medical Care Unit, Northwick Park Hospital, Harrow, Middlesex, UK. Submitted October 18,1990; accepted Februaly 19, 1991. Address reprint requests to Donogh P. O’Brien, PhD, Haemostasis Research Group, MRC Clinical Research Centre, Watford Rd, Harrow, Middlesex HA1 3UJ, UK. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1991 by The American Sociery of Hematology. 0006-497119117801-0014$3.0010 132 normal FVII. Also, the variant Wll was unable to compete with wild-type Wll for limited rabbit TF binding sites. A ligand blot procedure was used to directly demonstrate reduced binding of recombinanthuman TF to the variant FVll compared with normal FVII. Genetic analysis of leukocyte DNA showed a G to A mutation in the propositus’ gene at codon 304 that results in the substitution of a glutamine for an arginine residue in the catalytic domain of the protease. We conclude that this region of the Wll molecule is important for its function. o 1991 by The American Society of Hematology. MATERIALS AND METHODS Approval was obtained from the Ethics Committee of the Harrow Health District for these studies. The subject was informed that blood samples were obtained for research purposes only. Consent was given for the withdrawal of 500 mL of blood. His variant FVII was detected during the screening stage for the Thrombosis Prevention Trial: which includes FVII measurements. Monoclonal antibody (MoAb) to human FVII, RFF VII-2, was the gift of Dr A. Goodall (Royal Free Hospital, London, UK). MoAb to FVII 23117 was the gift of Dr F. Ofosu (McMaster University, Hamilton, Ontario, Canada). A7 MoAb to human FIX was the gift of Dr K. Smith (University of New Mexico School of Medicine, Albuquerque). Recombinant human TF apoprotein was the gift of Dr D. Higgins (Genentech Inc, South San Francisco, CA). For clotting assays this material was relipidated as described previously.’ Rabbit TF (thromboplastin) was from Sigma (Poole, Dorset, UK). Purified human FX was from Enzyme Research Laboratories (South Bend, IN). Sulpho-NHS biotin and streptavidin-horseradish peroxidase (HRP) were from Pierce Europe (Luton, UK). Prestained molecular weight standards for sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) were from BRL Uxbridge (Middlesex, UK). Superose 12 and TSK DEAE 5PW HPLC columns were from Pharmacia LKB (Milton Keynes, UK). The enhanced chemiluminescent detection reagent (ECL) was from Amersham International (Amersham, UK). Triton X-100 was from Calbiochem (San Diego, CA). All other reagents were from Sigma and were reagent grade or better. Protein estimations were performed by the method of Bradford“ using bovine serum albumin as standard. One-stage FVII coagulation assays were performed with immunodepleted FVII-deficient plasma as described previously.’ The coupled amidolytic assay for FVII was performed by the method of Seligsohn et a18: FVII antigen (FV1I:Ag) was measured using an enzyme-linked immunosorbent assay (ELISA) (Asserchrom FVI1:Ag; Diagnostica Stago, Asnieressur-Seine, France) according to the manufacturer’s instructions. FIX was purified according to the method of Smith’ from pooled normal human plasma donations. After elution from the antibody column, the peak FIX-containing fractions were concentrated and applied to a TSK DEAE 5PW column in 0.02 molL Tris HC1 pH 7.4 at a flow rate of 1mL/min. The column was then developed with a linear gradient from 0% to 75% buffer B (A + 1 molL NaCI). The purified protein was homogenous by SDS-PAGE and was stored at -70°C in 50% glycerol. For the biotinylation of coagulation factors, purified proteins were dialyzed into 100 mmol/L Na,HCO, pH 7.8. Ten micrograms of protein in a volume of 250 pL was biotinylated by the addition of Blood, Vol78, No 1 (July l), 1991:pp 132-140 From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 133 CHARACTERIZATIONOF FVll 304-GLN 10 mmol/L sulpho-NHS biotin. The mixture was incubated for 2 hours at room temperature, dialyzed against 0.15 mol/L NaCI, 0.05 mol/L Tris Hcl pH 7.4 (TBS), and stored at -20°C. Purification of FWZ. Five hundred milliliters of normal or patient blood was collected into 1/10 vol 3.8% trisodium citrateb mmol/L benzamidine hydrochloride and the plasma separated by centrifugation as described previously." BaCl,, 1 m o m , was then added dropwise to a final concentration of 50 mmoVL and the solution mixed for 1 hour on ice before centrifugation at 15,OOOg for 1 hour at 4°C. The precipitate was suspended in 50 mL 0.05 m o w Tris HCl pH 7.4 containing 0.15 mol/L NaCl (TBS) and recentrifuged. The pellet was then eluted in 50 mL TBS containing 0.2 m o m Na,EDTA. This solution was then dialyzed for 16 hours at 4°C against 0.02 mol/L Tris HCL pH 7.4 containing 0.25 mol/L NaCl (VI1 buffer). The dialysate was then applied at a flow rate of 30 mL/h to a 5 mL controlled pore glass-coupled RFFVII-2 antibody column equilibrated in FVII buffer. Five milligrams of protein A-purified IgG was coupled to this column as described previously." The column was linked in series with a Sepharose G25 guard column (PD 10; Pharmacia LKB). The column was then washed with 2 column vol of FVII buffer containing 1 mol/L NaCl and then developed isocratically with FVII buffer containing 1 mol/L sodium isothiocyanate adjusted to pH 8.5. Two-milliliter fractions were collected at a flow rate of 30 m u . Peak FVIIcontaining fractions were assayed by one-stage coagulation assay and then concentrated using Centricon-30 microconcentrators (Amicon, Danvers, MA). The concentrated eluate was then applied to a Superose 12 high performance liquid chromatography (HPLC) column, which was developed isocratically with TBS at a flow rate of 0.5 mumin. One-milliliter fractions were collected. Peak activity-containing fractions were pooled, reconcentrated, and stored in 50% glycerol at -70°C. Kinetic anatysis. Steady state kinetic parameters were derived for wild-type and CRM+ FVII using the modification of the methods of Seligsohn et aI8and Osterud et al.'* Briefly, 0.1 mL of CRM+ or normal diluted plasma containing 0.0125 U FV1I:Ag was added to 50 p L of a 1:5 dilution rabbit brain thromboplastin (Sigma) or relipidated human recombinant TF. Both of these TF preparations gave clot times of exactly 17 seconds in a prothrombin time assay using 20-normal pooled plasma. All proteins were diluted in TBS pH 7.4 containing 0.1% bovine serum album (TBSA). Purified FX diluted in TBSA was then added at various concentrations ranging from 300 to 37 nmol/L in a final volume of 50 pL. The mixture was incubated at 37°C for 1 minute at which time 25 pL 25 mmol/L CaC1, was added. Following a further incubation for 3 minutes at 37°C the reaction was stopped by the addition of 25 pL of 0.3 mol/L Na,EDTA. FXa generation was linear over this time period. TBSA, 600 JLL,was then placed in a cuvette with 100 pL of the chromogenic substrate S2222 (benzoylL-Ile-L-Glu-Gly-L-Arg-p-nitroanalide; Kabi-Vitrum, Uxbridge, UK) and 100 pL of the incubation mixture. The initial rate of change in absorbance at 405 nmoVL was then measured at 37°C in spectrophotometer (LKB). Results were represented as Lineweaver-Burk plots. Slopes and intercepts were calculated by linear regression analysis. All points were means of two experiments performed in duplicate. TF Zigand blots. FVII samples were electrophoresed on native 10%polyacrylamide slab gels. The monomer solution for these gels was 10% T 5% C. The separating gel buffer was 0.947 mol/L Tris HCl pH 8.48. The cathodic buffer was 37.6 m m o m Tris, 40 mmol/L glycine pH 8.89. The anodic chamber buffer was 63 mmol/L Tris, 50 mmol/L HCI, pH 7.47. No stacking gel was used, with combs being placed directly into the separating gel solution. Polymerization was effected by the addition of ammonium persulphate and N,N,N',N'-tetramethylethylenediamine. Samples were applied to the gels in 50% glycerol. Gels were run at room temperature at 37 mA constant current. After electrophoresis, gels were subjected to Western blotting at 4°C for 2 hours at 0.4 A onto nitrocellulose membranes. Membranes were blocked with NET/ TXlOO buffer (TBS, 0.1% Triton X-100,2.5 mmoVL CaCl,, 0.25% gelatin) for 1 hour at room temperature. Membranes were then incubated with 10 pL biotinylated recombinant human TF apoprotein in 15 mL NET buffer containing 0.1% Triton X-100 for 16 hours at room temperature. After washing with NETlTX100 for 1 hour the membranes were incubated for 15 minutes with 5 p L streptavidin-HRP complex in 15 mL of the same buffer, washed for 1 hour, and then exposed to XOMAT RP film (Eastman Kodak, Rochester, NY) following the addition of a chemiluminescent substrate (ECL-Amersham Ltd). Activation of FIX by wiZd-type and CRM+ W I . The activation of biotinylated FIX was assessed by cleavage of the single-chain zymogen to the two-chain enzyme in the presence of FVII and human or heterologous TF, as follows. Biotinylated FIX, 5 pL (0.2 pg), was added to 250 p L TBS containing 2.5 mmol/L CaCI, and 10 pL relipidated human or rabbit brain TF and 1 U unlabeled FIXa that was previously activated with purified human factor XIa bound to controlled pore glass." Twenty microliters was removed, acetone precipitated, and resuspended in 30 pL nonreducing sample buffer." Normal or CRM+ FVII, 0.1 U/FVII:Ag, was then added and samples were removed at various time points and acetoneprecipitated. Samples were subjected to SDS-PAGENestern blot analysis. Biotinylated FIX was detected using streptavidin-HRP and ECL detection system as described earlier. DNA isolation. DNA was isolated from lymphocyte pellets made from blood samples by established methods.13 In vitro amplification using the polymerase chain reaction (PCR). Exons 2 to 8 of the factor VI1 gene were amplified by PCR using the oligonucleotides listed in Table 1. Oligonucleotides were synthesized on an Applied Biosystems (Warrington, UK) 391 DNA synthesizer. One hundred nanograms of genomic DNA, 0.5 pg of each oligonucleotide, and 1.5 U Taq DNA Polymerase (Cetus, Beaconsfield, UK) were added to 90 p L buffer containing 1.5 mmol/L MgCl,, 10 mmol/L Tris-HC1 (pH 8.3), 50 mmol/L KCl, 0.01% gelatin, and 200 pmoVL each deoxynucleotide triphosphate. The reaction mixture was initially incubated at 94°C before undergoing 30 cycles of PCR using a Perkin-Elmer-Cetus Thermal Cycler (Beaconsfield, UK). PCR conditions are given in Table 1. DNA cloning and sequencing. The amplified fragments were gel purified and cloned into Gemini plasmid vectors (Promega Biotec, Southampton, UK) or Bluescript plasmid vectors (Stratagene, Cambridge, UK). The inserts were sequenced by the dideoxy chain termination m e t h ~ d . ' ~ RESULTS Measurement of FWZ antigen and activity. Table 2 shows the results of FVII antigen and activity assays performed on the plasmas of the CRM+ individual and his parents. All the individuals tested had normal FVI1:Ag levels as measured using an ELISA based on an HRP-conjugated polyclonal antibody to human FVII:Ag. Using rabbit brain TF in the one-stage FVII:C assay, both the parents showed reduced functional activity (ratio of FVII:C to FV1I:Ag 0.5:l) whereas the propositus had no detectable FVII clotting activity (ratio FVI1:C to FV1I:Ag 0:l). These data suggest that the parents are heterozygousfor the abnormal- From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 134 OBRIEN ET AL Table 1. Oligonucleotidesand Conditions Used to Amplify the Human Factor VI1 Gene Exon Oligonucleotides Denaturation 2 5'GCGGGGCACGCGGTGGGCGC3' 5'CGCCGCCGTGCAGTGTCCGC3' 94°C 45 s - 72°C 135s + 3 s 3+4 5'GGGTGCTCTGGTGAAGGTGC3' 5'CAGCTCCCCAGGGCCCTCTG3' 94°C 30 s 60°C 15 s 72°C 120 s 5 ~'CTGACCCCCAGIUGCCCCTC~' 5'CTAGTGGGACAGGGACTGGT3' 94°C 30 s 65°C 15 s 72°C 120 s 6 5'TAGTGGCACGTTCATCCCTC3' S'AAGGCTTCAAGACCCTCAGG3' 94°C 60 s 58°C 60 s 72°C 180 s 7 5'ATGACAGCAATGTGAClTCCO' 5'GTCTGTGGAAGTGACAGCAG3' 94°C 45 s 58°C 60 s 70°C 128 s 8 ~'GGCAGGTGGTGGIUAGGCCC~' 5'CCACAGGCCAGGGCTGCTGG3' 94°C 45 s 70°C 60 s 70°C 128 s ity and that the propositus is homozygous. Different results were obtained when human TF was used in the same assay. Significant FVI1:C levels of 0.3 U/mL were consistently recorded for the CRM+ individual. Identical results were obtained using relipidated recombinant human TF and TF from human brain. These data indicate that the CRM+ molecule is able to catalyze the conversion of FX and/or FIX to the active enzymes only in the presence of human TF and not with rabbit TF, in contrast to the wild-type molecule. Kinetics of FX activation by wild-type and CRM+ M Z . Experiments were conducted using a coupled amidolytic assay to derive steady-state kinetic parameters for wild-type or CRM+ FVII in the presence of human, bovine, or rabbit TF. In this assay the substrate, purified human FX, was varied in an initial incubation mixture in which the other reactants, FVII and TF, were held constant. After an initial incubation period the FXa generated was then added to a chromogenic substrate. The data are presented as Lineweaver-Burk plots where the reciprocal of the initial rate of generation of FXa (reciprocal of the initial AAbs/min-' of the chromogenic substrate) is plotted against the reciprocal of the substrate concentration (l/[FX] * pM-I). In experiments using human recombinant TF and normal FVII the Km for FX activation was 220 nmol/L, Vmax 0.052 AAbs/min-' (Fig 1). In the same experiment results using the CRM+ FVII were Km 2.6 pmol/L, Vmax 0.062 AAbs/ min-', representing a 12-fold increase in the Km for FX activation compared with the normal enzyme. Similar results were obtained using saturating concentrations of bovine TF in the same assay: wild-type FVII Km 250 nmol/L, Vmax 0.066 AAbdmin-', CRM+ FVII Km 2.2 bmol/L, Vmax 0.057 AAbs/min-'. Consistent with the Table 2. One-Stage FVII: C Assays Using Rabbit (rTF), Human (hTF), and ELlSAfor FVII:Ag Annealing Extension results obtained with the one-stage FVI1:C assays the CRM+ FVII molecule was unable to catalyze the conversion of FX to FXa in this assay in the presence of rabbit TF. To determine if the CRM+ FVII competed with normal FVII for rabbit TF binding sites the following experiments were performed: The concentration of rabbit TF was titrated in the amidolytic assay to a rate-limiting concentration and the steady-state kinetic parameters derived for normal FVII, Km 135 nmol/L, Vmax 0.107 AAbs/min-' (Fig 2A) The apparent decrease in the Km in this system reflects the reduced concentration of competing phospholipid in the reaction mixture as a consequence of the lower TF m bo 40 20 -m N I I : C U/mL -5 ; IO 5 I5 20 1 Subject rTF hTF FVII:Ag U/mL Father Mother Propositus 0.42 0.42 0.00 0.51 0.56 0.3 1.09 0.99 1.04 CFXI ( phi-') Fig 1. Lineweaver-Burk plot for the activation of FX by CRM+ and wild-type FVll in the presence of human TF. FVII, (-1; CRM+ FVII, (----). From www.bloodjournal.org by guest on October 28, 2014. For personal use only. CHARACTERIZATION OF FVll 304-GLN “ 1 A -I== I 135 ...e- I I C I I I 40 .*-_.-- ’ .--- _/- I -10 -5 0 1 I I I 5 IO 15 20 1 C Z 7 (uM-’) Fig 2. Lineweaver-Burk plot for the activation of FX by normal FVll in the presence of rabbit TF. (A) Normal FVll alone; (E) normal FVll with twofold molar excess of CRM+ FVII; (C) normal FVll with a fivefold molar excess of CRM+ FVII. concentration added. At higher T F concentrations the increased phospholipid competes with the substrate, FX.I5 Increasing amounts of the CRM+ FVII antigen were then added to the reaction mixture containing the normal FVII, rabbit TF, and FX. Km and Vmax for FVII activation of FX were unaltered in the presence of a twofold and a fivefold molar excess of the CRM+ FVII over the normal enzyme (Fig 2, B and C, respectively). These data indicate that the CRM+ molecule did not compete with the wild-type for the available TF binding sites in the reaction mixture. Purification and Western blot analysis of wild-type and CRM+ M I . Table 3 shows the results of the purification of CRM+ FVII from 190 mL of frozen citrated plasma. The protocol involved adsorption to barium chloride, affinity chromatography using the MoAb RFFVII 2, and Superose 12 HPLC gel permeation chromatography. Overall recoveries of normal FVII using this protocol were routinely 10% to 20%. The CRM+ and the wild-type molecules gave similar recoveries of FVI1:Ag at each step. Thus, the CRM+ molecule was not grossly different in size, charge, or posttranslational modification, and the epitope for the MoAb was present. Wild-type FVII purified in this manner Table 3. Purification of CRM+ FVll From Plasma FVII:Ag No. Vol (mL) FVII:C (U/mL) UlmL Total % Recovery Plasma BaC12 eluate RFFVll eluate Superose 12 190 190 15 1 <1 <1 <1 <1 1.04 2.5 5.3 33.3 197 107 80 30 100 98 40 15 FVII: C assays were performed using rabbit TF. has a specific activity of 1,500 U FVI1:C activity/mg by one-stage FV1I:C assay and coupled amidolytic assay: indicating that the molecule does not undergo activation as a consequence of the purification procedure. Western blot analysis under reducing conditions using a second MoAb as the probe (231/7) showed that the relative mobility was identical to the wild-type molecule (Fig 3). This result confirmed that there was no gross difference in relative molecular mass between the wild-type and the CRM+ molecule. Activation of purified FIX by CRM+ and normal FVII. Kinetic analysis demonstrated that the CRM+ FVII was capable of catalyzing the conversion of FX to FXa at a reduced rate in the presence of human, but not rabbit, TF. In addition, the CRM+ molecule was unable to compete with normal FVII for limited rabbit TF binding sites in the coupled amidolytic assay. To investigate the ability of the CRM+ molecule to proteolyze FIX, purified biotinylated human FIX was incubated at 37°C with either human or rabbit TF, calcium, CRM+ or normal FVII and a trace amount of cold FIXa to fully activate the FVII. Aliquots were removed at various time points and subjected to SDS-PAGE under nonreducing conditions. Gels were subsequently Western blotted onto nitrocellulose membranes, probed with streptavidin-HRP, and detected with a chemiluminescent substrate. Figure 4A shows the time-course activation of biotinylated human FIX in the presence of human TF and normal FVII (lanes 1 through 6) and CRM+ FVII (lanes 7 through 12). Rapid activation of the single-chain FIX, as evidenced by the appearance of the 43-Kd two-chain polypeptide, was seen in the reaction mixture containing wild-type FVII. Some proteolysis of the zymogen FIX was detected in the reaction mixture containing CRM+ FVII and human TF, but at a much slower rate and to a lesser extent than that seen with the wild-type molecule. In contrast, the same experiment performed using rabbit TF showed a rapid conversion of FIX to FIXa by the wild-type FVII but little or no conversion in the presence of the CRM+ enzyme (Fig 4B). After a 16-hour incubation of the FIX with the rabbit TF and the dysfunctional FVII, minimal activation was detected. These data confirm that while the CRM+ FVII was able to catalyze the conversion of FIX to FIXa in the presence of human TF at a reduced rate, no significant proteolytic activity could be detected in the presence of the rabbit cofactor. Ligand blot analysis. To investigate further the interaction between normal and CRM+ FVII and TF, a ligand blot procedure was developed. In this system purified FVII was subjected to nondenaturing polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membrane. The membranes were then blocked with a gelatin/ CaCl,/Triton X-100 containing buffer (NET buffer) and probed with nonrelipidated, biotinylated recombinant human TF apoprotein, in the presence of 0.1% Triton X-100. Binding of the biotinylated T F was detected using HRPconjugated streptavidin and a chemiluminescent detection system (ECL). In control experiments no binding of T F to purified FX, FVIII, FIX, or prothrombin could be detected and binding of TF to FVII was detected in the presence of From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 136 OBRIEN ET AL 200- 976843- 29- 1 Fig 3. Western blot analysis of normal (lane 1) and CRM+ (lane 2) FVll purtfied from plasma. Samples were electrophoresed in reduced SDS-PAGE 10% gels and Western blotted onto nitrocellulose and probed with MoAb 231/7 a FVll antibody labeled with '9. Relative molecular mass x 10' are shown on the left of the figure. 2 A 97- 6843Fig 4. SDS-PAGEIWestern blot analysis of the time course of the activation of labeled FIX by CRM+ and normal FVII. (A) Activation of FIX by normal FVll in the presence of human TF (lanes 1 through 6) and CRM+ FVII (lanes 7 through 12). Time points were 0,5 minutes, 30 minutes, 1 hour,2 hours, and 3 hours. (E) Activation of FIX by normal FVll in the presence of rabbit TF (lanes 1 through 8) and CRM+ FVll (lanes 9 through 16). Time points were 0,5 minutes, 30 minutes, 1 hour, 2 hours. 3 hours, 6 hours, and 16 hours after the addition of FVII. See Materials and Methods for full details. 26B 200- 43 97 68 26 - 1 2 3 4 5 6 7 8 9 10 11 12 From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 137 CHARACTERIZATION OF FVll 304-GLN calcium-containing buffers, but not in the presence of chelating agents such as EDTA (data not shown). Figure 5 shows the result of a ligand blot of normal and CRM+ FVII probed with recombinant biotinylated TF. An equal amount of FVII:Ag, as determined by ELISA, was loaded in each case. Significantlyreduced binding of the T F to the CRM+ molecule was detected (Fig 5A, lane 1) with respect to the wild-type molecule (Fig 5A, lane 2). Two lanes representing the same antigen loadings were run concurrently and subjected to Western blot analysis using an HRP-conjugated polyclonal antihuman FVII antibody as the probe (Fig 5B). This demonstrated that the amounts of FVII and CRM+ FVII loaded were similar. The upper band represents a small amount of residual FVII which remained in the top of the gel. This phenomenon was not always seen. In a separate experiment 1,0.5,0.25, and 0.125 U of normal or CRM+ FVII were electrophoresed and first probed with biotin-TF (Fig 6A) and reprobed with the HRP-conjugated polyclonal antibody to FVII (Fig 6B). Because the chemi-luminescent reaction inactivates HRPconjugated streptavidin it was possible to reprobe TF ligand blots with the polyclonal antibody to FVII to ensure that the same amount of FVII had been electrophoresed in each case. The results of the ligand blot analysis clearly demonstrate that the CRM+ FVII molecule exhibited reduced TF binding. Densitometric scans showed that the binding of recombinant human TF was approximately 10- to 15-fold lower that the binding to normal FVII. FUZgene analysis. To identify the mutation causing the observed phenotype, exons 2 through 8 were amplified by PCR. The amplified fragments were completely sequenced after cloning into plasmid vectors. The sequence of the cloned PCR products did not differ from the previously published sequence for the human FVII gene,I6 except at positions 10828 and 9778. The single-base change at 9778 is in intron 8 and therefore cannot be responsible for the altered phenotype of the FVII protein. The single-base B Fig 5. Ligand blot analysis of normal and CRM+ FVII. Samples were electrophoresed on 10% nondenaturing PAGE gels and Western blotted onto nitrocellulose membranes. (A) Purified CRM+ FVll (lane 1)and normal FVll (lane2) probedwith biotinylated human TF. (B) The same sample loads probed with an HRP-conjugated polyclonal antibody to human FVll (lane 1 CRM+ FVII, lane 2 normal FVII). See Materials and Methods for details. Fig 6. (A) Ligand blot analysis of normal FVll and CRM+ probed with biotinylated human TF. Lane 1, 1 U normal FVII; lane 2, 0.5 U; lane 3, 0.25 U; lane 4, 0.125 U; lane 5, 1 U CRM+ N 1 1 ;lane 6, 0.5 U; lane 7,0.25 U; lane 8,0.125 U. (B) The same membrane reprobedwith an HRP-conjugatedpolyclonal antibody to human FVII. change at position 10828results in a substitution of arginine by glutamine at position 304 (Fig 7). DISCUSSION In this study we have undertaken the biochemical analysis of a dysfunctional FVII molecule, isolated from the plasma of a clinically unaffected individual. A differential response to human and rabbit brain TF was detected in one-stage and amidolytic FVI1:C functional assays. Significant but reduced FVI1:C activity was detected in the presence of human TF, but no activity was detected in the presence of rabbit TF. Even at high concentrations of FVI1:Ag the CRM+ FVII was unable to catalyze the conversion of FX to FXa in the presence of the rabbit cofactor. In a coupled amidolytic assay using human TF and purified FX a 1Zfold increase in the Km for FX activation was recorded for the CRM+ over the wild-type enzyme, with no difference in the Vmax. These results may reflect the requirement of TF binding to FVII for substrate recognition, as described by Nemerson and Gentry.” These investigators have proposed an ordered addition, essential activation model of the T F pathway of coagulation, in which the enzyme (FVI1a)-activator (TF) complex picks up sub- From www.bloodjournal.org by guest on October 28, 2014. For personal use only. OBRIEN ET AL 138 G T /; 3' Thr A G T A G T Leu C *\; G Gln304 C pro 5' strate (FX or FIX) to form a ternary complex; a model that precludes the formation of binary enzyme-substrate complexes. Thus, in this model the affinity of the enzyme for its substrate is dependent on prior TFFVIIFlrIIa binding. It is also possible that, in addition, defects of substrate recognition or zymogen activation may result from the point mutation detected in this study. It has been shown that FVII bound to TF is more readily activated by FXa.18 Defective TF binding could conceivably result in reduced enzyme activation in the assays. The dysfunctional molecule did not compete with wild-type FVII for limited rabbit TF binding sites, because the addition of a fivefold molar excess of the CRM+ molecule to a reaction mixture containing wild-type FVII, a rate-limiting concentration of rabbit TF, and FX did not alter the kinetic parameters derived for the normal enzyme. This result indicated that the CRM+ molecule was unable to act as a competitive inhibitor of the wild-type FVII. To expand these studies we have developed a purification protocol that effects the purification of FVII from plasma donations of approximately 200 mL. FVII purified in this manner is isolated in zymogen form, as evidenced by a 1:l correlation between the results of the one-stage coagulation and amidolytic assays for FVII:C.8 Yields of FVII over the multistep process are routinely 10% to 20%. Purified CRM+ and normal FVII isolated from plasma by this protocol had identical relative mobilities on SDS-PAGE/ Western blot analysis, indicating that the defect did not arise from a major deletion or rearrangement of the gene. Using the purified CRM+ FVII we were able to demonstrate that the dysfunctional enzyme was able to proteolyze purified human FIX at a reduced rate in the presence of human, but not rabbit, TF. These data indicated that the defect in this molecule may involve the interaction with TF. Fig 7. Nucleotidesequence of exon 8 of the factor VI1 304-Gln gene. Exon 8 was sequenced by the dideoxy chain termination method (Sanger et al) after subcloning the amplified material into the Bluescript plasmid vector (Stratagene). Arrow indicates the nucleotide substitution. The translation of the sequence is shown. To confirm this hypothesis a ligand blot assay was developed to assess the binding of biotinylated recombinant human T F to membrane-bound normal and CRM+ FVII. This assay is specific for the T F W I I interaction because, in control experiments, there was no evidence of binding of biotinylated T F to related vitamin K-dependent coagulation factors. Biotinylated TF bound specifically to FVII in calcium-containing buffers but not in EDTA-containing buffers. The binding of TF-apoprotein to immobilized FVII is known to be calcium dependent." Therefore, the assay was used to investigate the binding of biotinylated recombinant human TF to purified wild-type and CRM+ FVII. With identical antigen loading the binding to the CRM+ molecule was repeatedly 10- to 15-fold less than that to the wild-type FVII. To characterize the genetic defect, exonic and flanking intronic sequences of the propositus' FVII gene were amplified, cloned, and sequenced by the dideoxy chain termination method.14 The only discrepancy with the previously published sequence of the human FVII gene'6 detected within the coding sequence of the CRM+ gene was a G to A substitution in codon 304, leading to the substitution of a glutamine for an arginine residue. This most probably is the result of a C to T transversion on the noncoding strand in a CpG dinucleotide. There is now a considerable body of evidence in the literature that CpG dinucleotides are mutation hot spots in the human genome.*' This mutation gives rise to a radical substitution because arginine side chains are positively charged at physiologic pH, whereas glutamine is a polar but uncharged residue. Little is known about the structural requirements of the FVII molecule for interaction with TF. The data presented in this study suggest that the arginine residue at position 304 (Arg-304), which is located in the catalytic domain of From www.bloodjournal.org by guest on October 28, 2014. For personal use only. CHARACTERIZATION OF FVll 304-GLN 139 the protease, may be important in this interaction. The substitution of Arg-304 by Gin-304 could prevent the association of the FVIIhabbit TF complex and allow only weak binding of the variant molecule to human TF. Presumably, differences in the primary structure of rabbit and human TF account for the differentia1 response Of the CRM+ FVI1 to the hUnan and rabbit cofactors*Rep1acement of Arg-304 by Gln-304 might cause a conformational change in the FVII molecule resulting in reduced TF binding affinity, through a propagat effect on a distal binding site for TF. Alternatively, the mutation may occur directly in the TF binding site. It is also possible that the mutation impacts more than one functional domain of the enzyme. The present study cannot distinguish between these possibilities. By comparison with the primary structures of the other vitamin K-dependent coagulation factors, it is evident that Arg-304 is located in a structurally conserved region of FVII. Furie et a1” have proposed an alignment of the bovine vitamin K-dependent coagulation factors based on the crystallographic coordinates of bovine trypsin and chymotrypsin. We have superimposed the sequences of the human zymogens on this alignment. Using this model FVII, Arg-304 is homologous to FIX Arg-333, FX Arg-298, prothrombin Arg-490, and protein C His-326 (Fig 8). This residue is not conserved in elastase, chymotrypsin, or trypsinogen. However, there is an insertion of three residues unique to FVII before a conserved cysteine residue immediately C-terminal to Arg-304, and a second insertion of five residues from positions 311 to 315 not found in the other proteases. Thus, while FVII Arg-304 is located in a region that is highly conserved in FIX, FX, prothrombin, and protein C, the insertions in FVII reduce the homology between it and the other vitamin K-dependent coagulation factors around this position. Furie et alZ1identified six variable regions and seven conserved regions in the bovine vitamin K-dependent coagulation factors. Using the same model FVII Arg-304 would form part of conserved region 5, which is not in or near the active site of the enzyme. Sequences N-terminal to conserved region 5 form part of variable region 4 which, it was speculated, may define a recognition domain for the binding of proteins to FIX, FX prothrombin, protein C, and therefore, by inference FVII. Interestingly, Arg to Gln-333 of FIX has been identified as the mutation in patients with severe hemophilia B (in whom there is synthesis and expression of CRM+ FIX).” FIX Arg-333 would be homologous to FVII Arg-304 according to the alignment proposed by Furie et al.” Because one w 0 dU!lo0 1 G0L LD nGA TA L R T HEKGR0STR L A , y G I( s. L L E I( EAKRNRTF L R V F H K G R S A L G0 p , L M v vv F K v G D s P N I T E Y M F c FPP .. S S I F I I T 0 N Y F C Fx . . . A T C . . . . . L R S T K F T I Y N N M F C FIX . . . . .@D s T R I R I T D N M F c I Iv E R . . , Pv VP H . . . N E . . . . .SEVMONMVS ENML Pc LN v P R t MToD P V DR P L V D R Y L E Y L R NL I K I P . . .NS C .dl LOOs R , . 0 Ifl Fig 8. Alignment of sequences of the human vitamin K-dependent coagulation factors based on the model proposed by Furie et al.” Dots depict areas where an insertion in another protease is found. The arginine residue at position 304 in FVll is marked with an asterisk (“1. consequence of the substitution of FVII Arg-304 to Gln is defective cofactor interaction it is possible that substitution of FIX Arg-333 to Gln results in an FIX molecule with defective FVIII binding. There are several lines of evidence to suggest that the cofactor binding sites of the vitamin K-dependent group of serine proteases reside in the catalytic domains of these molecules. An MoAb has been mapped to residues 180 to 310 of human FIX by Frazier et alz3and this antibody has been shown to interfere with the binding of FVIII to FIX.24 This region of FIX is homologous to the variable region of FVII N-terminal to the arginine at position 304. This would place the FVIII binding site on FIX close to the analogous region of FVII where the mutation described in this study leads to defective TF binding. The defect in FX Friuli, a CRM+ molecule with normal activation by R W X but defective activation by the intrinsic and extrinsic activators, has been attributed to abnormal cofactor (TF and FVIII) binding.” The precise location of the presumed point mutation in this variant FX gene has not been identified but the defect has been mapped to the catalytic domain of the molecule. Two recent reports also support the hypothesis that the FVII TF binding site resides in the catalytic domain of the molecule. Kumar et a126reported that a peptide spanning residues 206 to 218 of FVII inhibited FVII/TF interaction. In contrast, Wildgoose et a12’ were unable to demonstrate inhibition of FVII/TF interaction with this peptide but reported that three catalytic domain peptides, 195 to 206, 263 to 274, and 314 to 326 were inhibitory, with 195 to 206 inhibiting binding of human FVII to TF expressed on the surface of human bladder carcinoma (582) cells. In this report, using a combined epidemiologic and biochemical approach, another catalytic domain residue, Arg-304, has been shown to be important in FVII function. ACKNOWLEDGMENT We thank David Howarth for performing one-stage factor VI1 assays and Dr Yoshiaki Ohkubo for the purification of FIX. REFERENCES 1. Bajaj S , Rapaport S , Brown S : Isolation and characterization of human factor VII. J Biol Chem 256:253, 1981 2. Bach R, Gentry R, Nemerson Y FVII binding to tissue factor in reconstituted phospholipid vesicles: Induction of cooperativity by phosphatidylserine. Biochemistry 25:4007,1986 3. Nemerson Y Tissue factor and hemostasis. Blood 71:1, 1988 4. Meade Tw: Low-dose warfarin and low-dose aspirin in the primary prevention of ischemic heart disease. Am J Cardiol65:7C, 1990 5. O’Brien DP, Tate K, Giles A, Vehar G: FVIII-bypassing activity of bovine tissue factor using a canine hemophilic model. J Clin Invest 82:206, 1988 6. Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248,1976 7. Takase T, Tuddenham EGD, Chand S , Goodall AH: Monoclonal antibodies to factor VII: Production of immunodepleted plasma for FVII:C assays. J Clin Pathol41:342, 1988 From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 140 8. Seligsohn U, Osterud B, Rapaport S: Coupled amidolytic assay for factor VII: Its use with a clotting assay to determine the activity status of factor VII. Blood 52:978,1978 9. Smith KJ: Immunoaffinity purification of factor IX from commercial concentrates and infusion studies in animals. Blood 721269,1988 10. O’Brien DP, Tuddenham EDG: Purification and characterization of FVIII 1689-cys:A nonfunctional cofactor occurring in a patient with severe haemophilia A. Blood 73:2117,1989 11. Ohkubo Y, OBrien DP, Kanehiro T, Fukui H, Tuddenham EGD: Characterisation of a panel of monoclonal antibodies to human coagulation factor XI and detection of FXI in HepG2 cell conditioned medium. Thromb Haemost 63:3, 1990 12. Osterud B, Kasper CK, Lavine KK, Prodanos C, Rappaport SI: Purification and properties of an abnormal blood coagulation factor IX (factor IX BM). Thromb Haemost 45:55,1981 13. Kunkel LM, Smith KD, Boyer SH, Borgaonkar DS, Wachtel SS, Miller OJ, Breg WR, Jones HW, Rary JM: Analysis of human Y chromosome-specific reiterated DNA in chromosome variants. Proc Natl Acad Sci USA 74:1245,1977 14. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463, 1977 15. Bom V, Reinalda-Poot J, Cupers R, Bertina RM: Extrinsic activation of human blood coagulation factors IX and X. Thromb Haemost 63:224,1990 16. O’Hara P, Grant F, Haldeman B, Gray C, Insley M, Hagen F, Murray M: Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation. Proc Natl Acad Sci USA 84:5158,1987 17. Nemerson Y, Gentry R: An ordered addition, essential activation model of the tissue factor of coagulation: Evidence for a conformational case. Biochemistry 25:4020,1986 OBRIEN ET AL 18. Rao L, Rapaport S: Activation of factor VI1 bound to tissue factor: A key early step in the tissue factor pathway of blood coagulation. Proc Natl Acad Sci USA 85:6687, 1988 19. Broze G, Leykam J, Schwartz B, Miletich J: Purification of human brain tissue factor. J Biol Chem 260:10917, 1985 20. Gitschier J, Kogan S, Levinson B, Tuddenham EGD: Mutations of factor VI11 cleavage sites in hemophilia A. Blood 72:1022, 1988 21. Furie B, Bing D, Feldman R, Robison D, Burnier J, Furie BC: Computer generated models of blood coagulation factor Xa factor IXa and thrombin based upon structural homology with other serine proteases. J Biol Chem 257:3875,1982 22. Gitschier F, Green PM, High KA, Lozier JN, Lillicrap D, Ludwig M, Olek K, Reitsma P, Goosens M, Yoshioka A, Sommer S, Brownlee G: Haemophilia B: Database of point mutations and short additions and deletions. Nucleic Acids Res 18:4053,1990 23. Frazier D, Smith K, Cheung W, Ware J, Lin S, Thompson A, Reisner H, Bajaj S, Stafford D: Mapping of monoclonal antibodies to human FIX. Blood 74:971,1989 24. Bajaj S, Rapaport SI, Maki S: A monoclonal antibody to factor IX that inhibits the factor VI1I:Ca potentiation of factor X activation. J Biol Chem 260:11574,1985 25. Fair D, Revak D, Hubbard J, Girolami A Isolation and characterization of the factor X Friuli variant. Blood 73:2108, 1989 26. Kumar A, Blumenthal D, Fair D: Identification of regions of factor VI1 mediating the assembly of the extrinsic pathway activation complex. Blood 74:95a, 1989 (abstr, suppl) 27. Wildgoose P, Kazim A, Kisiel W: The importance of residues 195-206 of human blood clotting factor VI1 in the interaction of factor VI1 with tissue factor. Proc Natl Acad Sci USA 87:7290,1990
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